Name: a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants
Uploaded: 2016-01-01T14:00:00
Description: Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com

This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

Note: The following protocol describes the SPE performed on 100 ml water sample with C18 sorbent and elution with 50% v/v methanol. The enriched sample is diluted to reach 10% v/v methanol before analysis with an enzyme linked immunosorbent assay (ELISA) kit.
1. Preparing the Reagents
<ol>
	<li>Prepare the water samples
	<ol>
		<li>Prior to any other step, filter each 100 ml water sample with 0.2 &#181;m pore size filters.</li>
		<li>Spike the sample with desired concentration by diluting ap...

<ol>
	<li>Chang, H. S., Choo, K. H., Choi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methods+of+identification,+analysis+and+removal+of+endocrine+disrupting+compounds+(EDCs)+in+water.">The methods of identification, analysis and removal of endocrine disrupting compounds (EDCs) in water.</a> J. Hazard. Matter. 172, 1-12 (2009).</li><li>Azzouz, A., Souhail, B., Ballesteros, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+solid-phase+extraction+and+gas+chromatography-mass+spectrometry+determination+of+pharmaceuticals+and+hormones+in+water+samples.">Continuous solid-phase extraction and gas chromatography-mass spectrometry determination of pharmaceuticals and hormones in water samples.</a> J. Chromatogr. A. 1217, 2956-2963 (2010).</li><li>Tomsikova, H., Aufartova, J., Solich, P., Novakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+analysis+of+female-steroid+hormones+in+environ-mental+samples.">High sensitivity analysis of female-steroid hormones in environ-mental samples.</a> Trends Anal. Chem. 34, 35-58 (2012).</li><li>Ciofi, L., Fibbi, D., Chiuminatto, U., Coppini, E., Checchini, L., Del Bubba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+on-line+solid+phase+extraction+coupled+to+high-performance+liquid+chromatography-tandem+mass+spectrometric+analysis+at+sub-ng/L+levels+of+selected+estrogens+in+surface+water+and+wastewater.">Fully automated on-line solid phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometric analysis at sub-ng/L levels of selected estrogens in surface water and wastewater.</a> J. Chromatogr. A. 1283, 53-61 (2013).</li><li>Robles-Molina, J., Lara-Ortega, F. J., Gilbert-Lopez, B., Garcia-Reyes, J. F., Molina-Diaz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-residue+method+for+the+determination+of+over+400+priority+and+emerging+pollutants+in+water+and+wastewater+by+solid-phase+extraction+and+liquid+chromatography-time-of-flight+mass+spectrometry.">Multi-residue method for the determination of over 400 priority and emerging pollutants in water and wastewater by solid-phase extraction and liquid chromatography-time-of-flight mass spectrometry.</a> J. Chromatogr. A. 1350, 30-43 (2014).</li><li>Huang, C. H., Sedlak, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+estrogenic+hormones+in+municipal+waste+water+effluent+and+surface+water+using+enzyme-linked+immunosorbent+assay+and+gas+chromatography/tandem+mass+spectrometry.">Analysis of estrogenic hormones in municipal waste water effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry.</a> Environ. Toxicol. Chem. 20, 133-139 (2001).</li><li>Hintemann, T., Schneider, C., Sch&#246;ler, H. F., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+study+using+two+immunoassays+for+the+determination+of+estradiol+and+ethinylestradiol+in+the+aquatic+environment.">Field study using two immunoassays for the determination of estradiol and ethinylestradiol in the aquatic environment.</a> Water Res. 40, 2287-2294 (2006).</li><li>Farr&#233;, M., Kuster, M., Brix, R., Rubio, F., Lopez de Alda, M. J., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+an+estradiol+enzyme-linked+immunosorbent+assay+kit,+liquid+chromatography-tandem+mass+spectrometry,+and+ultra-performance+liquid+chromatography-quadrupole+time+of+flight+mass+spectrometry+for+part-per-trillion+analysis+of+estrogens+in+water+samples.">Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography-tandem mass spectrometry, and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples.</a> J. Chromatogr. A. 1160, 166-175 (2007).</li><li>Pu, C., Wu, Y. F., Yang, H., Deng, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+analysis+of+contraceptive+drug+levonorgestrel+in+waste+water+samples+by+a+newly+developed+indirect+competitive+enzyme-linked+immunosorbent+assay+(ELISA)+coupled+with+solid+phase+extraction.">Trace analysis of contraceptive drug levonorgestrel in waste water samples by a newly developed indirect competitive enzyme-linked immunosorbent assay (ELISA) coupled with solid phase extraction.</a> Anal. Chim. Acta. 628, 73-79 (2008).</li><li>Van Emon, J. M., Gerlach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+status+report+on+field-portable+immunoassay.">A status report on field-portable immunoassay.</a> Environ. Sci. Technol. 29 (7), 312A-317A (1995).</li><li>Farr&#233;, M., Brix, R., Barcel&#242;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+water+for+pollutants+using+biological+techniques+under+European+Union+funding+during+the+last+10+years.">Screening water for pollutants using biological techniques under European Union funding during the last 10 years.</a> Trends Anal. Chem. 24 (6), 532-545 (2005).</li><li>Schneider, C., Sch&#246;ler, H. F., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+enzyme-linked+immunosorbent+assay+for+ethinylestradiol+using+a+long-chain+biotinylated+EE2+derivative.">A novel enzyme-linked immunosorbent assay for ethinylestradiol using a long-chain biotinylated EE2 derivative.</a> Steroids. 69, 245-253 (2004).</li><li>Heub, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+and+portable+solid+phase+extraction+platform+for+immuno-detection+of+17&#946;-estradiol+in+water.">Automated and portable solid phase extraction platform for immuno-detection of 17&#946;-estradiol in water.</a> J. Chrom. A. 1381, 22-28 (2015).</li><li>Petrovic, M., Eljarrat, E., Lopez de Alda, M. J., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+disrupting+compounds+and+other+merging+contaminants+in+the+environment:+a+new+survey+on+new+monitoring+strategies+and+occurrence+data.">Endocrine disrupting compounds and other merging contaminants in the environment: a new survey on new monitoring strategies and occurrence data.</a> Anal. Bioanal. Chem. 378, 549-562 (2004).</li><li>Richardson, S. D., Ternes, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+analysis:+Emerging+contaminants+and+current+issues.">Water analysis: Emerging contaminants and current issues.</a> Anal. Chem. 86, 2813-2848 (2014).</li></ol>

A new method for the preparation of water samples followed by analysis using immunoassay was proposed. The instrument enables to perform solid phase extraction in an automated and user-friendly way.
The filtration of the water sample prior to its injection into the system is critical. Any particulates still present in the solution would potentially cause clogging of the fluidic network and obstruct the SPE column. Another important step is the preparation of the SPE column. The amount of parti...

Sample preparation is an important step in any analytical process. In particular, removal of matrix effects, diminution of interferences, and enrichment of the analyte are necessary to obtain precise results and reach low limits of detection. Endocrine disrupting compounds (EDCs) are of particular concern due to their action on the living organisms even when present at very low levels in the environment. The natural hormone 17&#946;-estradiol is present on the EU water pollution Watch List and prone to be added to the list of priority substances regulated under the European Water Framework Directive. Solid phase extraction (SPE) is commonly applied for the analysis of small pollutants in water, with both chemical 1-5 (chromatography, mass spectrometry) and immunological 6-9 detection methods. The latter gained interest in the field of environmental monitoring, as immunoassays are available in large variety of formats, are specific to the target analyte, and reach low limits of detection.6, 7, 10, 11 Various enzyme linked immunosorbent assays (ELISA) are commercially available and enable to analyze multiple samples at once on a multi-well plate. The procedure consists in successive reaction steps that can take a few hours. The final product of reaction can be detected optically to determine the concentration of the target molecule based on a calibration curve.
Classical SPE procedures include sorbent pre-conditioning, sample extraction, washing, elution, and concentration by evaporation of the eluent. The solvent used for dilution of this extract is chosen depending on the detection method. For immunological methods, the amount of organic solvent influences strongly the sensitivity of the method.12
In addition to the recovery and the pre-concentration performances, the method also needs to be simple and cost efficient. Automation of the procedure helps to reduce human-related errors. In our previous work 13 we introduced our prototype for automated SPE, and our method was applied to the analysis of the natural hormone 17&#946;-estradiol in sea water samples. With the present video we would like to highlight the technical advantages of our method compared to traditional off-line and on-line SPE, and its particular compatibility with detection by immuno-reactions. We describe the protocol applied to water samples for the detection of 17&#946;-estradiol. SPE is performed with octadecyl-silica (C18) sorbent phase and elution is performed with diluted methanol.

<table border="0" cellpadding="0" cellspacing="0" width="609">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Filter membrane 0.2 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">GNWP04700</td>
			<td style="width:167px;">For sample filtration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon membrane 11 &#956;m pore size</td>
			<td style="width:107px;">Merck Millipore</td>
			<td style="width:119px;">NY1104700</td>
			<td>For SPE column</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable biopsy punch 5 mm</td>
			<td style="width:107px;">Medical Budget</td>
			<td style="width:119px;">39302439</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nucleodur C18 ec&#160;</td>
			<td style="width:107px;">Macherey Nagel</td>
			<td style="width:119px;">713550.01</td>
			<td>50 &#956;m particle diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic sea water</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">SSWS500-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol standard</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;"></td>
			<td>300 ng/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17beta-estradiol ELISA kit&#160;</td>
			<td style="width:107px;">Enzo Life Science</td>
			<td style="width:119px;">ADI-900-008</td>
			<td>96 wells, range 30 - 3000 ng/L</td>
		</tr>
	</tbody>
</table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

Reproducibility of sorbent packing was evaluated by drying and weighting the pipetted sorbent in glass vials and the result is shown in Figure 1. Reproducibility of the time of injection was tested for 100 ml samples, as shown in Figure 2. Concentration in initial and pre-concentrated spiked samples were determined by using a commercial ELISA kit for 17&#946;-estradiol and are shown in Figure 3.
<p class="jove_content" fo:keep-togethe...

Watch this Scientific Journal Video about A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants at JoVE.com 

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants (Video) | JoVE 

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Research

JoVE Journal

Environment

A Small Volume Procedure for Viral Concentration from Water

A Small Volume Procedure for Viral Concentration from Water (Video) | JoVE 

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples (Video) | JoVE 

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies (Video) | JoVE 

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples (Video) | JoVE 

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA (Video) | JoVE 

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples (Video) | JoVE 

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis (Video) | JoVE 

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs (Video) | JoVE 

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA (Video) | JoVE 

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method (Video) | JoVE 

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...